Cytotoxic bispecific monoclonal antibody, its production and use

ABSTRACT

The present invention discloses, a bispecific monoclonal antibody to an ansamitocin derivative and a target antigen, particularly tumor-associated antigen, which can carry an ansamitocin derivative in a stable and inactive form at other sites than the target and release the active-form ansamitocin derivative at the target site, so that an anticancer agent having excellent durability and selectivity with little adverse action can be prepared using the bispecific monoclonal antibody and ansamitocin derivatives.

This is a continuation of copending application Ser. No. 07/457,343filed on Dec. 27, 1989, now U.S. Pat. No. 5,141,730.

FIELD OF THE INVENTION

The present invention relates to a bispecific hybrid monoclonalantibody. More specifically, the present invention relates to a hybridmonoclonal antibody (hereinafter also referred to as hybrid MoAb) whichis bispecific to an ansamitocin derivative and a target antigen,particularly tumor-associated antigen on the surface membrane of acancer cell, and to a polydoma which produces it.

The present invention also relates to a method of cancer treatment inwhich the above-mentioned MoAb is used to specifically bind anansamitocin derivative to a cancer cell, killing the cancer cell.

DESCRIPTION OF THE PRIOR ART

Ansamitocin (hereinafter also abbreviated ANS), a drug possessing potentantitumor activity, was discovered in fermentation products of anactinomycete (genus Nocardia) [E. Higashide et al.: Nature, 270, 721(1977)]. Although an ansamitocin analog, maytansine (hereinafter alsoabbreviated MAY), had already been isolated as a plant-derived antitumorsubstance [S. M. Kupchan et al.: Journal of the American ChemicalSociety, 94, 1354 (1972)], much attention was given to ANS, which isderived from bacteria, since the yield of MAY is poor. Both of the drugshave an action mechanism similar to that of vinca alkaloid drugs [e.g.vincristine (hereinafter also abbreviated VCR)], inhibiting theformation of microtubules in tumor cells, thus having a cytocidaleffect. The cytotoxicity of ansamitocin is 10 to 100 times as potent asthat of conventional anticancer chemotherapeutic drugs (e.g.methotrexate, daunomycin). Ansamitocin and its derivatives, as a newtype of antitumor agent, have been synthesized by chemical modificationsand microbial conversions [A. Kawai et al.: Chemical and PharmaceuticalBulletin, 32, 2194 (1984); H. Akimoto et al.: Chemical andPharmaceutical Bulletin, 32, 2565 (1984); A. Kawai et al.: Chemical andPharmaceutical Bulletin, 32, 3441 (1984)]. Through studies on theantitumor effects of these ansamitocin derivatives, in vitro and invivo, using MAY and VCR as control drugs, as well as subacute toxicitystudies including digestive tract disorder and neurotoxicity studies,several kinds of ansamitocin derivatives were found to be superior toMAY and VCR in terms of effect and safety. In particular,9-thiomaytansine, represented by the following structural formula,showed a superior chemotherapy coefficient value (toxic amount/effectiveamount) to MAY and VCR. ##STR1##

However, ANS due to its potent cytotoxicity can cause adverse effects.As a result, ANS is now in the same situation as was MAY, thedevelopment of which was stopped due to digestive tract disorder andneurotoxic action.

On the other hand, what are called `missile therapy drugs,` namelyantitumor immunocomplexes prepared by binding an antitumor antibody to achemotherapeutic agent or a biotoxin, were developed as drugs thatselectively kill tumor cells. They are characteristic of recognizing andbinding to a tumor-specific antigen or tumor-associated antigen on tumorcells, and inhibiting the DNA synthesis, protein synthesis, ormicrotubule formation, in these tumor cells to kill them. Therefore,they are specific to tumorous organs, tissues and cells, and have littleadverse action on normal cells. Some antibody-drug or antibody-toxincomplexes have already been clinically applied, with some favorableresults. However, the antibody activity or the pharmacological activityof the drug or toxin is often reduced due to the chemical bindingreaction between the antibody and the drug or toxin protein; it isexpected that more potent selective antitumor agents will be developed.

In particular, in using low molecular weight antitumor agents, therehave been several problems; for example, 1 an appropriate functionaLgroup must be used for binding to an antibody, 2 chemical binding to anantibody produces a greater effect on the activity than that of a toxicprotein, and normally the pharmacological activity is reduced to 1/10 to1/100 or less, and thus 3 particular conditions must be provided underwhich the linker portion of the antibody-drug complex can be cleaved intumor cells.

With this technical background, the present inventors conductedinvestigations to develop a new type of antibody-drug complex by bindingANS, which possesses potent cytotoxicity, to an anticancer antibody tomake it tumor specific. In the preparation of such an antibody-drugcomplex, some problems were considered to arise as stated above. Theproblems posed by the conventional method utilizing chemical bindingreaction include 1 remarkable reduction of the antitumor activity of thedrug, as well as reduction of the binding activity of the antibody tothe antigen, 2 byproduction of homopolymers formed between antibodiesthat are inert as selective antitumor agents or of high molecularpolymers not useful in vivo due to easy metabolizability, 3 difficultyin controlling the number of drug molecules binding to the antibody,making it impossible to obtain an antibody-drug complex of constantquality, and 4 difficulty in utilizing a drug having the besttherapeutic coefficient value due to the limited number of drugs usablefor antibody binding to those having an appropriate functional group.

Also, recently developed hybridoma preparation techniques have permittedthe preparation of new types of triomas, tetraomas and other polydomas[C. Milstein et al.: Nature, 305, 537 (1983); M. R. Suresh et al.:Proceedings of the National Academy of Science, U.S.A., 83, 7989(1986)], thus making it possible to prepare bispecific antibodies havingnew functions [Japanese Unexamined Patent Publication No. 12276/1988(Hybritech); U.S. Pat. No. 4,714,681 (University of Texas System CancerCenter)].

SUMMARY OF THE INVENTION

The present inventors conducted investigations of application andextension of the new technology described above to solve these problems.As a result, the present inventors succeeded in developing a hybrid MoAbthat does not necessitate chemical binding procedures between antibodyand drug molecule, which had been essential in the preparation ofconventional antibody-drug complexes, and that permits preparation of anantibody-drug immunocomplex retaining the entire bioactivities of theantibody and the drug. The present inventors then prepared ananti-human-cancer protein complex using the hybrid MoAb. Accordingly,the present invention relates to a polydoma which produces a bispecifichybrid MoAb specific to both ansamitocin derivatives and targetantigens, e.g. tumor-associated antigens on cancer cell membranes.

The present invention also relates to a bispecific hybrid MoAb producedby a tetraoma. This tetraoma is obtained by fusing a hybridoma, whichproduces an antibody against ansamitocin derivatives (hereinafter alsoreferred to as anti-ANS antibody) and another hybridoma, which producesan antibody against target antigens, for example, human transferrinreceptor (hereinafter also abbreviated hTfR), which is often expressedon cancer cell membranes. The present invention further relates to aselective anti-human-cancer protein complex obtained using this hybridMoAb.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the preparation of the polydoma that produces the bispecific hybridMoAb of the present invention, an anti-ANS-antibody-producing hybridomais used, which can, for example, be prepared by the method describedbelow.

An ansamitocin derivative is first inoculated into an animal to elicitthe production of anti-ANS antibody. In this case, the use of ANS aloneas an immunogen does not normally induce the production of antibody withhigh titer; therefore, an ansamitocin derivative having an appropriatefunctional group is used as an immunogen, in conjunction with thecarrier protein such as bovine serum albumin (hereinafter alsoabbreviated BSA) or thyroglobulin. The carrier protein can be complexedwith an ansamitocin derivative via the appropriate functional group, forexample, the carboxyl group of PDM-3-C₂₀ -carboxymethyl ether or theamino group of maytansinol 3-α-aminophenylacetate or PDM-3-C₂₀-p-aminobenzyl ether, represented by the following formulas.

    ______________________________________                                         ##STR2##                                                                     Compound R              Y            Q                                        ______________________________________                                        PDM-3-C.sub.20 -                                                                       COCH(CH.sub.3).sub.2                                                                         CH.sub.2 COOH                                                                              OH                                       carboxy-                                                                      methyl ether                                                                  Maytansinol                                                                            COCH(NH.sub.2)C.sub.6 H.sub.5                                                                CH.sub.3     OH                                       3-α-amino-                                                              phenylacetate                                                                 PDM-3-C.sub.20 -                                                                       COCH(CH.sub.3).sub.2                                                                         CH.sub.2 (C.sub.6 H.sub.4)NH.sub.2                                                         OH                                       p-amino-                                                                      benzyl ether                                                                  9-Thiomay- tansine                                                                      ##STR3##      CH.sub.3     SH                                       MAY                                                                                     ##STR4##      CH.sub.3     OH                                       ANS      COCH(CH.sub.3).sub.2                                                                         CH.sub.3     OH                                       ______________________________________                                          Note: PDM3 represents 20demethoxy-20-hydroxymaytansinol 3isobutyrate.   

Examples of subject animals for inoculation include rabbits, rats, miceand guinea pigs; it is especially preferable to use mice for MoAbproduction.

Inoculation can be achieved by an ordinary method. For example, theimmunogen, in an amount of 1 to 100 μg, preferably 10 to 25 μg perinoculation, is emulsified with 0.1 ml of physiological saline and 0.1ml of Freund's complete adjuvant, and then is inoculated, subcutaneouslyinto the back or intraperitoneally into the abdomen, 3 to 6 times duringa 2 to 3 week period.

From the group of immunized animals, mice for example, those having highantibody titer are selected. Spleens or lymph nodes are collected 3 to 5days after the final immunization; antibody-producing cells containedtherein are fused with myeloma cells. Cell fusion can be conducted inaccordance with a known method. Examples of the fusogen includepolyethylene glycol (hereinafter abbreviated PEG) and Sendai virus; itis preferable to use PEG. Examples of the myeloma cell line includeNS-1, P3U1 and SP2/0; it is preferable to use P3U1. It is preferablethat the ratio of, for example, splenocytes and myeloma cells, is 1:1 to10:1. It is recommended that PEG with a molecular weight of 1,000 to9,000 be added at a concentration of 10 to 80%, and that incubation beconducted at 20° to 37° C., for 3 to 10 minutes.

Various methods can be used for the screening ofanti-ANS-antibody-producing hybridomas, including the ELISA method. Inthe ELISA method the culture supernatant of hybridomas is added to amicroplate to which a (maytansinol 3-α-aminophenylacetate)-human serumalbumin (hereinafter also abbreviated HSA) complex is adsorbed; next, ananti-mouse immunoglobulin antibody labeled with horseradish peroxidase(HRP) is added to the microplate, and the anti-ANS monoclonal antibodybound to the solid phase plate is then detected. Hybridomas positive forantibody activity, are selected and bred on a medium supplemented withHAT (hypoxanthine-aminopterin-thymidine). These hybridomas areimmediately subjected to cloning; this cloning is normally easilyachieved by, for example, limiting dilution method or other means. Theantibody titer of the culture supernatants of the cloned hybridomas isdetermined by the above-mentioned method, to select hybridomas whichstably produce an antibody with high titer. As a result, using theabove-described production method, the desired monoclonalanti-ANS-antibody-producing hybridoma can be obtained.

Examples of hybridomas that are produced in accordance with theproduction method above and that produce anti-ANS antibody (IgG₁, λchain) include mouse hybridoma AS6-44.9.

Examples of the tumor-associated antigen as the target antigen includehTfR, which is relatively highly expressed in various tumor cell lines.hTfR can be purified from human placenta tissue in accordance with aknown method [P.A. Seligman et al.: Journal of Biological Chemistry,254, 9943 (1979)]. A sample of hTfR with high purity is normallyobtained by the method described below. 1 Human placenta tissue ishomogenized in a phosphate-buffered saline (20 mM disodium phosphate,0.15M NaCl; hereinafter also abbreviated PBS), pH 7.5, containing 4%Triton-X-100, followed by sonication and centrifugation. 2 The resultingsupernatant, after being subjected to salting-out with ammonium sulfate,is applied to a column coupled with an antibody against humantransferrin (hTf) and thoroughly washed with a phosphate buffer (20 mMdisodium phosphate, hereinafter also abbreviated PB), pH 7.5, containing0.5M NaCl, followed by elution of hTfR fraction with a 0.02M glycinebuffer (pH 10.0) containing 0.5M NaCl and 0.5% Triton-X-100. 3 Theobtained hTfR fraction is applied to a hTf-coupled column. After thecolumn is washed with PB (pH 7.5) containing 1M NaCl, elution isconducted using a 0.5M glycine buffer (pH 10.0) containing 1M NaCl and1% Triton-X-100 to yield a purified sample of hTfR. A single stepelution and isolation is also possible using a column coupled withanti-hTfR antibody.

Animal immunization with hTfR and cell fusion ofanti-hTfR-antibody-producing cells with myeloma cells can be conductedin the same manner as described for ansamitocin derivatives. Variousmethods can be used for the screening of anti-hTfR-antibody-producinghybridomas. Examples of such methods include the ELISA method and thecell-ELISA method. In the ELISA method the culture supernatant of thehybridomas is added to a microplate to which anti-mouse IgG antibody isadsorbed, and the purified sample of hTfR labeled with HRP is thenadded. The anti-hTfR monoclonal antibody bound to the solid phase plateis subsequently detected. In the cell-ELISA method, K562 cell strainwhich expresses a large amount of TfR on their surface membrane, isimmobilized on a microplate, and the culture supernatant of hybridomasis added to the microplate, and then HRP-labeled anti-mouse IgG antibodyis added.

Examples of hybridomas that are prepared in accordance with theabove-mentioned production methods and produce anti-hTfR antibody (IgG1,k chain) include mouse hybridoma 22C6.

There are several methods of preparing the polydoma of the presentinvention, which produces a bispecific hybrid MoAb [H. Aramoto et al.:Proteins, Nucleic Acids and Enzymes, 33, 217 (1988)]; any method can beused. Example of such methods include 1 the method in which theabove-mentioned HAT-resistant, anti-ANS-antibody-producing hybridoma isacclimated step-by-step to a medium supplemented with5-bromodeoxyuridine (hereinafter also abbreviated BrdU), followed bycloning of a thymidine kinase deficient strain and making it HATsensitive; similarly, an HAT-resistant anti-hTfR-antibody-producinghybridoma is made resistant to 8-azaguanine (hereinafter alsoabbreviated AZG), followed by cloning ofhypoxanthine-guanine-phosphoribosyl transferase deficient strain andmaking it HAT sensitive; these two strains are then fused in accordancewith a conventional method to yield tetraomas, which are then cultivatedon an HAT-supplemented medium for selection, followed by cloning of atetraoma which secretes a hybrid antibody possessing a binding activityboth to ansamitocin derivatives and to hTfR; and 2 the method in whichan anti-ANS-antibody-producing hybridoma is labeled with fluoresceinisothiocyanate (hereinafter also abbreviated FITC); ananti-hTfR-antibody-producing hybridoma is labeled with tetramethylrhodamine isothiocyanate (hereinafter also abbreviated TRITC); these twolabeled hybridomas are fused in accordance with a conventional method;the obtained cell suspension is applied to a fluorescein-activated cellsorter (hereinafter also abbreviated FACS) to select and clone atetraoma emitting both the green fluoresence of FITC and red fluoresenceof TRITC. The markers for the parent strains can be reversedly used inselecting and cloning the desired tetraoma.

For cell fusion in these procedures, a fusogen such as Sendai virus orPEG, electric stimulation or other means is used. Among others, PEG ispreferable. An example of the use of PEG is described below, but thepresent invention should not be limited to this method. PEG having amolecular weight of about 1,000 to 9,000 is used at a concentration ofabout 10 to 80%; treating time is about 0.5 to 30 minutes. Efficientfusion can be achieved by keeping about 35 to 55% PEG 6000 in contactwith cells at 37° C. for 4 to 10 minutes; these conditions arepreferable.

Polydoma selection can be achieved in the above-mentionedHAT-supplemented medium and other media; for this purpose, the drugacclimation method using 8-AZG, 6-thioguanine, 5-BrdU or other drug canbe used to obtain strains resistant to the drug. Introduction of a newmarker into fused cells permits the use of various selection media.Examples of such selection media include neomycin-supplemented mediumand hygromycin B-supplemented medium [B. Sugden et al.: Molecular andCellular Biology, 5, 410 (1985)]. Also available is the method in whichtwo hybridomas labeled with different fluorescent dyes are fused,followed by sorting double-labeled hybrid hybridomas by FACS, asdescribed above [L. Karawajew et al.: Journal of Immunological Methods,96, 265 (1987)].

Various methods can be used for the screening ofhybrid-antibody-producing polydomas. Examples of such methods include 1the above-mentioned ELISA method for the screening ofanti-ANS-antibody-producing hybridomas; 2 the ELISA method in which theculture supernatant of polydoma is added to a solid phase microplate towhich anti-mouse immunoglobulin antibody is adsorbed, and HRP-labeledhTfR is then added to detect the anti-hTfR antibody bound to the solidphase plate; 3 the ELISA method in which the culture supernatant isadded to a solid phase microplate to which a (maytansinol3-α-aminophenylacetate)-HSA complex is adsorbed, and HRP-labeled hTfR isthen added to detect the bispecific hybrid antibody; and, when using ananti-hTfR antibody (K chain) having a light chain different from that ofanti-ANS antibody (λ chain), 4 the ELISA method in which the culturesupernatant is added to a solid phase microplate to which a (maytansinol3-α-aminophenylacetate)-HSA complex is adsorbed, and HRP-orbiotin-labeled anti-mouse IgG-_(K) chain specific antibody is then addedto detect the bispecific antibody, and modifications of these methods;these methods can also be used in combination as appropriate.

Polydomas positive for hybrid antibody activity are subjected tocloning, which can normally be carried out easily by the limitingdilution method or other methods. The culture supernatant of the clonedpolydomas is subjected to antibody titer determination by theabove-mentioned method to select the polydoma that stably produces anantibody with high titer. As a result, using the methods describedabove, the desired hybrid monoclonal antibody-producing polydoma can beobtained.

Cultivation of the above-mentioned polydoma of the present invention cannormally be carried out in a liquid medium or in the peritoneal cavityof animals (e.g. in the peritoneal cavity of mammals such as mice) by aknown method. Purification of the antibody from the culture broth orascites fluid is carried out by using a combination of known biochemicaltechniques. For example, the cell culture broth or ascites fluid iscentrifuged; the resulting supernatant is collected and subjected tosalting-out (normally using ammonium sulfate or sodium sulfate). Theobtained protein precipitate is dissolved in an appropriate solution anddialyzed, and subjected to column chromatography (using, e.g., an ionexchange column, gel filtration column, Protein A column, hydroxyapatitecolumn) to separate and purify the desired antibody. Single stepseparation and purification can also be conducted by the process using acolumn in which two different antigens have been immobilized.

The separation and purification procedures described above can yield,for example, about 1 to 5 mg of hybrid MoAb with purity not less than80% by protein weight from 1 l of the culture supernatant. From 20 ml ofthe ascites fluid, 3 to 10 mg of the same antibody can be obtained.

The hybrid MoAb thus obtained is a uniform protein, and, for example,F(ab')₂ fragments retaining a binding activity both to ansamitocinderivatives and to cancer-associated antigens, such as hTfR, can beobtained by proteinase treatment; these fragments can serve for the samepurpose as the hybrid MoAb of the present invention.

Examples of hybrid-antibody-producing polydomas prepared in accordancewith the above-mentioned production method include tetraoma ATF1-170,described in Example 2 below.

An example of the polydoma that produces the hybrid MoAb of the presentinvention, is the tetraoma formed between an anti-ANS MoAb-producinghybridoma and an anti-hTfR MoAb-producing hybridoma, mentioned above. Itshould be noted, however, that a trioma formed between a hybridoma whichproduces one of the MoAbs and a cell which produces the other MoAb or ahybridoma obtained by cell fusion of two cells which produce respectiveMoAb species after immortalization using Epstein-Barr virus or othermeans can serve the same purpose as the above-mentioned tetraoma, aslong as they produce the hybrid MoAb of the present invention.

Moreover, in cases where these polydomas produce mouse IgG MoAb, it ispossible to prepare a mouse-human chimeric antibody by obtaining DNAwhich encodes a variable region containing the antigen recognition siteof the bispecific hybrid MoAb and ligating a gene which encodes theconstant region of human IgG thereto using a gene manipulation technique[Z. Steplewski et al.: Proceedings of the National Academy of Science,USA, 85, 4852 (1988)]. This chimeric antibody is advantageously used foradministration to humans because of its low antigenicity.

The bispecific antibody of the present invention, or ananti-human-cancer protein complex prepared from an ansamitocinderivative and the bispecific antibody, can be used as treatment methodsin cancer therapy. Examples of such methods include 1 the method inwhich the hybrid MoAb of the present invention is administered to thecancer-bearing patient and an ansamitocin derivative is administeredafter a sufficient length of time to ensure its binding to cancer tissueor cells; and 2 the method in which the hybrid MoAb and an ansamitocinderivative are administered to the cancer-bearing patientsimultaneously; but preferably is used 3 the method in which the hybridMoAb and an ansamitocin derivative are reacted, and the unreactedportion of the ansamitocin derivative is separated, and then theresulting anti-human-cancer protein complex is administered to thecancer-bearing patient. In this case, any ansamitocin derivative can beused, as long as it possesses antitumor activity and is capable ofreacting with the anti-ANS antibody. Examples of such ansamitocinderivatives include compounds represented by the following formula:##STR5## [wherein R represents a hydrogen atom or an acyl group derivedfrom a carboxylic acid; Q represents a hydroxyl group (OH) or a mercaptogroup (SH); X represents a chlorine atom or a hydrogen atom; Yrepresents a hydrogen atom, a lower alkylsulfonyl group, or an alkylgroup or an aralkyl group, either of which may have a substituent], and4,5-deoxy derivatives thereof.

Examples of the acyl group derived from a carboxylic acid represented byR in the above formula (I) include acyl groups derived from carboxylicacids having a molecular weight of not greater than 300 or acyl groupshaving 1 to 20 carbon atoms. Examples of such acyl groups includesaturated or unsaturated aliphatic acyl groups, saturated or unsaturatedalicyclic acyl groups, aromatic acyl groups, and N-acyl-α-amino acidtype acyl groups; they can be represented by, for example, the followingformula:

    --COR.sup.1                                                (A)

[wherein R¹ represents a hydrogen atom, an alkyl group, an alkenylgroup, a cycloalkyl group or an aryl group; these groups may have asubstituent; the cyclic group above may bind to the carbonyl group viaan alkylene chain]. As an example in which a substituent is contained,there is mentioned an N-acyl-α-aminoacyl group represented by theformula: ##STR6## [wherein R² represents a hydrogen atom, an alkylgroup, a cycloalkyl group or an aryl group; these groups may have asubstituent; the cyclic group may bind to the carbon atom at theα-position via an alkylene chain; R³ represents a hydrogen atom, analkyl group, a cycloalkyl group or an aryl group; these groups may havea substituent and the cyclic group may bind to the N atom via analkylene chain; R⁴ represents a hydrogen atom, an alkyl group, analkenyl group, a cycloalkyl group or an aryl group; these groups mayhave a substituent and the cyclic group may bind to the carbonyl groupon the N atom via an alkylene chain; R⁴ may represent an alkoxy group ora benzyloxy group].

R¹ in the acyl group represented by the above formula (A) is hereafterdescribed in detail.

Examples of the alkyl group represented by R¹ include alkyl groupshaving about 1 to 18 carbon atoms (e.g. methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,1-methylpropyl, hexyl, heptyl, 3-heptyl, octyl, nonyl, decyl, undecyl,dodecyl, tridecyl, pentadecyl and heptadecyl groups). Among others, alower alkyl group having about 1 to 6 carbon atoms is preferable.

Examples of the alkenyl group represented by R¹ include alkenyl groupshaving about 2 to 10 carbon atoms (e.g. vinyl, allyl, 1-methyl-vinyl,2-methyl-vinyl, 1-octenyl and 1-decenyl groups). Among others, a loweralkenyl group having about 2 to 4 carbon atoms is preferable.

Examples of the cycloalkyl group represented by R¹ include cycloalkylgroups having about 3 to 10 carbon atoms (e.g. cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, nonybornyl andadamantyl groups).

Examples of the aryl group represented by R¹ include a phenyl group anda naphthyl group. A phenyl group is preferable.

The alkyl group, the alkenyl group, the cycloalkyl group and the arylgroup as R¹ may have a substituent. Examples of the substituent includelower alkoxy groups having 1 to 4 carbon atoms (e.g. methoxy, ethoxy,propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxygroups), lower alkanoyl groups having 2 to 4 carbon atoms (e.g. acetyl,propionyl, butyryl and isobutyryl groups), lower alkanoyloxy groupshaving 2 to 4 carbon atoms (e.g. acetyloxy, propionyloxy, butyryloxy andisobutyryloxy groups), lower alkoxycarbonyl groups having 2 to 4 carbonatoms (e.g. methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl andisopropoxycarbonyl groups), halogen atoms (e.g. chlorine, fluorine,bromine and iodine atoms), hydroxyl groups, nitro groups, cyano groups,trifluoromethyl groups, amino groups, mono-lower (C₁₋₄) alkylaminogroups (e.g. methylamino group), di-lower (C₁₋₄) alkylamino groups (e.g.dimethylamino, diethylamino, dipropylamino, diisopropylamino anddibutylamino groups), lower alkylthio groups having 1 to 4 carbon atoms(e.g. methylthio, ethylthio, propylthio, isopropylthio, butylthio,isobutylthio, sec-butylthio and tert-butylthio groups), lower (C₁₋₄)alkylsulfinyl groups, lower (C₁₋₄) alkanesulfonyl groups, oxo groups,thioxo groups and lower alkanoylamino groups having 1 to 4 carbon atoms(e.g. formylamino, acetylamino, propionylamino, butyrylamino andisobutyrylamino groups). When R¹ above is a cyclic group (cycloalkyl oraryl group), examples of the substituents also include lower alkylgroups having 1 to 4 carbon atoms (e.g. methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl and tert-butyl groups). The groupsmay be substituted by 1 to 3 members of the same or different species ofthe substituents.

The cyclic group (cycloalkyl or aryl group which may have a substituent)represented by R¹ may bind to the carbonyl group in the formula --COR¹via an alkylene chain. Examples of the alkylene chain include straightor branched lower alkylene chains having about 1 to 4 carbon atoms (e.g.methylene, ethylene, methylmethylene (ethylidene), propylene, butylene,1-,2- or 3-methylpropylene, 1- or 2-ethylethylene, propylmethylene, 1,1-or 1,2-dimethylethylene, isopropylmethylene). The alkylene chain mayhave a substituent which is mentioned above. Accordingly, when thecyclic group and the alkylene chain bind together, R¹ represents acycloalkylalkyl group or an aralkyl group which may have a substituent.

Examples of the alkyl group represented by R¹ having 1 to 18 carbonatoms and a substituent include methoxymethyl, butoxymethyl,methylthiomethyl, methylthioethyl, ethylthioethyl, isopropylthioethyl,butylthioethyl, isobutylthioethyl, acetyloxymethyl, acetyloxyethyl,ethoxycarbonylmethyl, butoxycarbonylethyl, fluoromethyl, chloromethyl,chloroethyl, 3-chloropropyl, 4-chlorobutyl, trifluoromethyl,bromomethyl, 4-bromobutyl, 5-bromopentyl, iodomethyl, 2-iodoethyl,cyanomethyl, methylsulfinylethyl and methylsulfonylmethyl.

Examples of the alkenyl group represented by R¹ having 2 to 10 carbonatoms and a substituent include 2-ethoxycarbonylvinyl.

Examples of the cycloalkyl group represented by R¹ having 3 to 10 carbonatoms and a substituent include 2,2-dimethylcyclopropyl,4-isobutylcyclohexyl, 2-bromocyclopropyl, 2-chlorocyclobutyl,4-chlorocyclohexyl, 2,2-difluorocyclobutyl, 3-methoxycyclohexyl,4-acetycyclohexyl, 2-cyanocyclobutyl, 4-cyanocyclohexyl and4-dimethylaminocyclohexyl.

Examples of the aryl group represented by R¹ having a substituentinclude 2-,3- or 4-methylphenyl, 4-tert-butylphenyl, 2-,3- or4-chlorophenyl, 2-,3- or 4-bromophenyl, 2-,3- or 4-iodophenyl, 2-,3- or4-fluorophenyl, 2- or 4-methoxyphenyl, 4-butoxyphenyl,4-methoxycarbonylphenyl, 3-acetylphenyl, 2-,3- or 4-nitrophenyl, 3- or4-cyanophenyl, 4-dimethylaminophenyl, 4-diethylaminophenyl,4-acetoxyphenyl, 4-butyryloxyphenyl, 3,4-dimethoxyphenyl,3,4,5-trimethoxyphenyl, 3,4-methylenedioxyphenyl,3-trifluoromethylphenyl, 4-methylthiophenyl, 4-methylsulfonylphenyl and4-acetamidophenyl.

When the cyclic group represented by R¹ described above [e.g. cycloalkyland aryl (particularly phenyl) groups] binds to the carbonyl carbon ofthe acyl group in the formula (A) via an alkylene chain, R¹ essentiallyrepresents a group comprising one of these cyclic groups and an alkylenechain bound thereto, for example, a cycloalkylalkyl group or an aralkylgroup. Examples of the cycloalkylalkyl group include adamantylmethyl,cyclohexylmethyl, 3-cyclohexylpropyl, 2-cyclopentenylmethyl and2-cyclopentylethyl. Examples of the aralkyl group include 4-bromobenzyl,2-,3- or 4-cyclobenzyl, 2,5- or 3,4-dimethoxybenzyl, 4-ethoxybenzyl,4-fluorobenzyl, 3- or 4-methoxybenzyl, 4-methoxyphenylethyl, 1- or2-naphthylmethyl, 2-,3- or 4-nitrobenzyl, 3-nitrophenethyl, benzyl,2-,3- or 4-phenylpropyl, 2-,3- or 4-methylbenzyl, 3,4,5-trimethoxybenzyland α-methylphenethyl.

The N-acyl-α-aminoacyl group represented by the above formula (B) isdescribed below.

The alkyl, alkenyl, cycloalkyl and aryl groups defined for R², R³ or R⁴are exemplified by the same groups as those mentioned as examples for R¹above. These groups may have a substituent. The substituent isexemplified by the same groups as those mentioned as examples for thesubstituent for R¹ above. The cyclic group for R², R³ or R⁴ (i.e.cycloalkyl or aryl group) may bind to the carbon atom at the α-position,the N atom or the carbonyl group attaching to the N atom in the formula(B) via an alkylene chain. The alkylene chain is exemplified by the samealkylene chains as those described referring to R¹ above.

Examples of the alkoxy group represented by R⁴ include lower alkoxygroups having about 1 to 4 carbon atoms (e.g. methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy groups).

Representative examples of the N-acyl-α-aminoacyl group represented bythe formula (B) include N-acetyl-N-methyl-glycyl,N-benzoyl-N-methyl-glycyl, N-(4-chlorobenzoyl)-N-methyl-glycyl,N-acetyl-N-methyl-alanyl, N-acetyl-N-benzyl-alanyl,N-acetyl-N-methyl-leucyl, N-isobutyryl-N-methyl-alanyl,N-isovaleryl-N-methyl-alanyl, N-propionyl-N-methyl-alanyl,N-acetyl-N-methyl-phenylalanyl,2-(N-acetyl-N-methyl)amino-3-methoxycarbonylpropionyl,2-(N-acetyl-N-methyl)amino-3-methylmercaptopropionyl,2-(N-acetyl-N-methyl)amino-3-ethylmercaptopropionyl,N-acetyl-N-methyl-isoleucyl, N-acetyl-N-methyl-leucyl,N-acetyl-N-methyl-methionyl, N-acetyl-N-methyl-phenylalanyl,N-acetyl-N-methyl-4'-acetoxy-tyrosinyl, N-benzyl-N-methyl-valyl,N-acetyl-N-methyl-phenylglycyl, N-acetyl-N-methyl-3-cyanoalanyl andN-acetyl-N-methyl-(4'-dimethylamino)-phenylalanyl.

Examples of the lower alkylsulfonyl group represented by Y in the aboveformula (I) include alkylsulfonyl groups having about 1 to 4 carbonatoms (e.g. methanesulfonyl, ethanesulfonyl, 2-propanesulfonyl,2-butanesulfonyl).

Examples of the alkyl group represented by Y include lower alkyl groupshaving about 1 to 8 carbon atoms (e.g. methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, pentyl, isopentyl, hexyl, heptyl, octyl). Examples ofthe aralkyl group include phenyl-lower (C₁₋₄) alkyl groups (e.g. benzyl,2-phenethyl, 3-phenylpropyl). The alkyl group and the aralkyl group forY may have a substituent. Examples of the substituent include hydroxylgroups, amino groups, lower (C₁₋₄) acylamino groups, lower (C₁₋₄)alkyloxy groups, benzyloxy groups, oxo groups, halogen (chlorine,bromine, iodine) atoms, trifluoromethyl groups, lower (C₂₋₅)alkoxycarbonyl groups, carboxyl groups, methylenedioxy groups and lower(C₁₋₄) alkylthio groups.

Examples of the corresponding 4,5-deoxy derivatives include compoundsrepresented by the formula: ##STR7## [wherein the symbols are as definedabove].

The above-mentioned ansamitocin derivatives can be synthesized by, forexample, the methods described in Kupchan et al., the Journal of theAmerican Chemical Society, 97, 5294 (1975), Higashide et al., Nature,270, 271 (1977), U.S. Pat. Nos. 4,137,230, 4,151,042, 4,162,940,4,228,239, 4,229,533, 4,248,870, 4,256,746, 4,260,608, 4,263,294,4,264,596, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269,4,309,428, 4,317,821, 4,322,348, 4,331,598, 4,356,265, 4,362,663,4,371,533 and 4,424,219 or similar methods thereto.

The target antigen in the present invention includes various antigens;representative examples include cancer cell membrane surface antigenssuch as tumor-associated antigens, immunocompetent cell surfacereceptors and virus infected cell surface antigens. Among theseantigens, hTfR is often used as the tumor-associated antigen, butcarcinoembryonic antigen (what is called CEA), α-fetoprotein and severalcancer-associated sugar chain antigens including CA19-9 [S. Hakomori:Cancer Research, 45, 2405 (1985)], B-cell lymphoma membraneimmunoglobulin idiotypes [R.A. Miller et al.: New England Journal ofMedicine, 306, 517 (1982)], T-cell lymphoma receptor idiotypes [L.L.Lanier et al.: Journal of Immunology, 137, 2286 (1986)] and glycoproteinwhich is expressed specifically on renal cell carcinoma are also usable.

As mentioned above, the hybrid MoAb of the present invention is capableof very specific binding to the target antigen and efficiently killingcancer cells by the cytotoxic action of the ansamitocin derivative boundthereto, thus permitting selective and effective cancer treatment.

The hybrid MoAb of the present invention neutralizes the cytotoxicity ofan ansamitocin derivative by binding with the ansamitocin derivative,and releases the ansamitocin derivative at the target site to producethe cytotoxic effects. Thus, the hybrid MoAb of the present inventioncan carry an ansamitocin derivative in a stable and inactive form atother sites than the target site and release the active-form ansamitocinderivative at the target site. As a result, the present inventionprovides an anticancer agent having excellent durability andselectivity, with very little adverse action.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the antibody dilution curve obtained by subjecting theanti-ANS antibody AS6-44.9 prepared in Example 1 to the ELISA methoddescribed in Reference Example 1.

FIG. 2 shows the neutralizing activity curve of the same AS6-44.9antibody against ANS (1 ng/ml).

FIG. 3 shows the bispecific antibody activity of the hybrid antibodyATF1-170 prepared in Example 2.

FIG. 4 shows the hydroxyapatite column elution curve of theimmunoglobulin species produced by the tetraoma ATF1-170 prepared inExample 3, on the basis of absorbance at 280 nm.

FIG. 5 shows the results of cytotoxicity test of anti-ANS-anti-hTfRbispecific antibody prepared in Example 3.

FIG. 6 shows the antibody dilution curve of the culture supernatant ofRCAS1-488.

The present invention is hereinafter described in more detail by meansof reference and working examples; these examples are not to beconstrued as limitations on the scope of the invention.

    ______________________________________                                        Deposition of animal cells at deposition institutions                                           IFO        FRI                                              Animal cell line  (IFO No.)  (FERM No.)                                       ______________________________________                                        Mouse hybridoma AS6-44.9                                                                        50181      BP-2233                                          Mouse-mouse hybridoma 22C6                                                                      50172      BP-2054                                          Mouse hybridoma ATF1-170                                                                        50182      BP-2234                                          Mouse hybridoma RCS-1                                                                           50184      BP-2333                                          Mouse hybridoma RCAS1-488                                                                       50218      BP-2687                                          ______________________________________                                         IFO: Institute for Fermentation, Osaka                                        FRI: Fermentation Research Institute, Agency of Industrial Science and        Technology, Ministry of International Trade and Industry Brief Descriptio     of The Drawings                                                          

REFERENCE EXAMPLE 1 ELISA for anti-ANS antibody assay

1 Preparation of solid phase antigen

Maytansinol 3-α-aminophenylacetate maleimidated withN-(γ-maleimido-butyryloxy)-succinimide was added to HSA which had beensubjected to modification reduction with N-succinimidylpyridyldithiopropionate to yield a (maytansinol 3-α-aminophenylacetate)-HSAcomplex by thiol exchange reaction. To a 96-well microplate was added a50 μg/ml solution of this protein complex at 100 μl/well to yield asolid phase antigen.

2 Assay method

To the above antigen-sensitized plate was added 100 μl of the subjectculture supernatant of hybridoma, followed by reaction at roomtemperature for 2 hours. After thorough washing of the plate with a 20mM phosphate buffered saline solution containing 0.05% Tween 20 (pH 7.3,hereinafter abbreviated PBS-Tw), an HRP-labeled anti-mouse IgG rabbitantibody was added, and reaction was carried out at room temperature for2 hours.

After the plate was washed again, a 0.1M citrate buffer solutioncontaining ortho-phenylenediamine and H₂ O₂ as enzyme substrates wasadded to each well, and enzyme reaction was carried out at roomtemperature. After termination of the reaction by the addition of 1Nsulfuric acid, the amount of coloring pigment was determined at awavelength of 492 nm using a Multiscan (produced by Flow Co.).

REFERENCE EXAMPLE 2 Preparation of anti-hTfR-antibody-producinghybridoma

1 Purification of hTfR

1.5 kg of human placenta tissue was cut into small pieces and blended inPBS (pH 7.5), followed by centrifugation. The resulting sediment washomogenized in PBS containing 4% Triton X-100. This homogenate wasultrasonicated and then centrifuged. To the resulting supernatant wasadded ammonium sulfate at about 32 g per 100 ml supernatant. Aftersalting-out, this mixture was applied to a column coupled with anti-hTfantibody, followed by thorough washing with PB (pH 7.5) containing 0.5MNaCl. The hTfR fraction eluted with a 0.02M glycine buffer solution (pH10.0) containing 0.5M NaCl and 0.5% Triton X-100 was applied to anhTf-coupled column. After the column was washed with PB containing 1MNaCl, elution was conducted using a 0.05M glycine buffer solution (pH10.0) containing 1M NaCl and 1% Triton X-100 to yield about 1.5 mg of apurified sample of hTfR.

2 Immunization

To a 200 μg/ml solution of the above purified sample of hTfR inphysiological saline was added an equal volume of Freund's completeadjuvant, followed by thorough emulsification. The resulting emulsionwas then administered intraperitoneally and subcutaneously at the backto BALB/c mice (female, n=10, 20 μg/ml/mouse). Additional immunizationwas conducted at intervals of 3 weeks. The animal that showed themaximum serum antibody titer 2 weeks after 4 additional immunizationswas intravenously given the same hTfR antigen solution as specifiedabove (30 μg/0.1 ml physiological saline/mouse).

3 Cell fusion

3 days after the final immunization, the spleen was excised and asplenocyte suspension was prepared by a conventional method(approximately 10⁸ cells). To this suspension was added 2×10⁷ mousemyeloma cells (P3U1), followed by cell fusion using PEG6000 inaccordance with the method of Kohler and Milstein [Nature, 256, 495(1975)].

After completion of cell fusion, the cell mixture was suspended in HATmedium containing hypoxanthine, aminopterin and thymidine, followed bycultivation for 10 days. After selection of parent cells, cultivationwas continued using HT medium which had the same composition as that ofHAT medium but not including aminopterin.

4 Selection and cloning of hypridomas

A commercially available anti-mouse IgG rabbit antibody solution (20μg/ml) was dispensed to a 96-well microplate at 100 μl per well. Afterthis microplate was allowed to stand at 4° C. overnight, PBS (pH 7.3)containing 2% BSA was added to prepare a sensitized plate. The purifiedsample of hTfR obtained in 1, after being labeled with HRP in accordancewith a conventional method, was used for ELISA [T. Kitagawa: Yuki GoseiKagaku, 42, 283 (1984)]. Accordingly, the culture supernatant ofhybridomas was added to the above second antibody-sensitized plate, andreaction was carried out at room temperature for 2 hours. After theplate was washed with PBS, HRP-labeled hTfR was added, followed byreaction at room temperature for 2 hours. Enzyme reaction was thencarried out by the method described in Reference Example 1-2, todetermine the antibody titer.

The hybridoma showing especially high binding activity was subjected tocloning by limiting dilution method to yieldanti-hTfR-antibody-producing hybridoma 22C6. The present antibody wasidentified as the IgG₁ (_(k) chain) subclass, exhibiting high affinityto human tumor cell strain K562.

REFERENCE EXAMPLE 3 Mixed hemagglutination assay, MHA

Among the subject cells, adherent cells (500 cells/well) were dispensedto a 60-well microplate (Nunc) and cultivated for 24 to 48 hours, whilenon-adherent cells were suspended in a medium with no serum, dispensedto wells (500 cells/well) and centrifuged at 400×g for 5 minutes foradhesion of the cells to the plate.

The indicator blood cells were prepared as follows: Sheep red bloodcells were washed with PBS 3 times and suspended in PBS to prepare a 2%suspension. The suspension was reacted with the same volume of mouseanti-sheep red blood cell antibody (Ortho) which was previously 2.5-folddiluted on maximum agglutination titer with PBS, at 37° C. for 30minutes. The blood cells were washed with PBS 3 times and suspendedagain in PBS in a concentration of 2%. The suspension was reacted withthe same volume of rabbit anti-mouse IgG antibody (Cappel) which waspreviously 25-fold diluted with PBS, at 37° C. for 30 minutes. The bloodcells were then washed with PBS 3 times and preserved as a 2%suspension.

The cell-adhering plate was washed with 0.1M MgCl₂ -0.03M CaCl₂ -0.1%glucose-containing Veronal buffered saline (pH 7.4, hereinafterabbreviated VBS) which further contained 5% FCS. A culture supernatantor ascites fluid was dispensed to each well of the plate and allowed tostand at room temperature for 1 hour. The plate was washed with VBS, andthen the indicator blood cell suspension which was diluted with 5%FCS-VBS to 0.2% was dispensed to each well and allowed to stand at roomtemperature for 40 minutes. The plate was washed with VBS to removeunreacted blood cells and then observed by a microscope. In the controltest, in which no antibody was added, a rosette was formed in notgreater than 1% of the cells. A "positive" test was defined as a rosetteformed by not less than 25% of the subjected cells.

REFERENCE EXAMPLE 4 Preparation of anti-human renal cell carinomamonoclonal antibody-producing hybridoma

1 Transplantation of human renal cancer cell and immunization by sera

A nu/nu-BALB/c mouse was subcutanously given a tumor tissue graft (2 mmsquare) from a patient with renal cancer to obtain a well-establishedrenal cancer cell AM-RC-3, which was then subcutanously transplanted toa syngeneic nu/nu-BALB/c mouse. After 3 to 4 weeks, sera were collected.A syngeneic BALB/c mouse was intraperitoneally given 0.5 ml of the serawith the same volume of Freund's complete adjuvant 6 times at 7- to10-day intervals, and then the mouse was intraperitoneally given 1.0 mlof the sera (final immunization). After the final immunization theantibody titer was determined by the MHA method described in ReferenceExample 3.

2 Preparation of hybridoma

Spleen cells of the immunized mouse which exhibited a high antibodytiter were fused with mouse myeloma cells, NS-1, according to aconventional method (treatment with PEG6000 at 37° C. for 1 to 10minutes), and hybridomas were selected using HAT medium (1×10⁻⁴ Mhypoxanthine, 4×10⁻⁷ M aminopterin and 1.6×10⁻⁵ M thymidine). Growinghybridoma groups were screened by the MHA method described in ReferenceExample 3. The group exhibiting a high antibody titer was further clonedto obtain antihuman renal cancer carinoma MoAb-producing mouse hybridomaRCS-1. RCS-1 antibody produced by mouse hybridoma RCS-1 was identifiedas the IgG₁ subclass.

3 Production of mouse MoAb

An MCH (AF)-nu mouse was intraperitoneally given 5×10⁶ mouse hybridomaRCS-1 cells. After about 4 weeks, 5 to 10 ml of ascites fluid wascollected. The collected ascites fluid was subjected to salting-out withammonium sulfate and then purification by a column of DEAE-cellulose.About 200 mg of the purified mouse anti-human renal cell carcinoma MoAbRCS-1 was obtained from 50 ml of ascites fluid.

4 Characteristics of mouse anti-human renal cell carcinoma MoAb

The reactivity of mouse MoAb RCS-1 with various human tumor cells andnormal renal tissues was determined using the MHA method described inReference Example 3. The results are shown in the following Table.

The table clearly shows that the antibody has strong reactivity to allkinds of the subjected renal cancer cells while it is not reacted withnormal renal tissues. Further, the antibody is reactive with a part oflung cancer cells, bladder cancer cells and T-cell leukemia cells.

Table Reactivity of mouse monoclonal antibody RCS-1¹) Positive cellgroups

Renal cancer (AM-RC-3, AM-RC-6, AM-RC-7, SK-RC-1, SK-RC-9, SK-RC-18)

Bladder cancer (T-24)

Lung cancer (Luci-10, Calu-6, PC-10)

T-cell leukemia (HUT-78)

Negative cell groups

Bladder cancer (KK-47, MGH-U-1)

Prostate cancer (DU-145)

Stomach cancer (NUGC-2, NUGC-3, NUGC-4, MKN-28, KATO-III, MRK-1)

Intestine cancer (SW-403, SW-620, SW-1116, SW-1222, CaOV-4, HT-29)

Uterocervical cancer (ME-180)

Melanoma (SK-MEL-33, SK-MEL-37)

Breast cancer (MCF-7)

Glioma (MG-178)

Lung cancer (ADLC-DA, SBC-3, SCLC-SA, Luci-6, CADO-LC3, OKADA, QG-56)

T-cell leukemia (CCRF-CEM, HPB-ALL, HSB-2, HUT-102, RPMI-8402,

P12/Ichikawa, MT-1, MT-2)

B-cell leukemia (Raji, Daudi, BALL-1, RPMI-1788, Ly-16)

Null cell leukemia (NALL-1, NALM-6, NALM-18, KOPN-K, P30/Ohkubo)

Myelocytic leukemia (HL-60)

Negative tissue groups

Normal kidney (5 kinds)

1): Determined by the MHA method of Reference Example 3

EXAMPLE 1 Preparation of anti-ANS-antibody-producing hybridoma andimmunization

PDM-3-C₂₀ -carboxymethyl ether was converted to an active ester byN-hydroxysuccinimide and dicyclohexylcarbodiimide and then bound to thecarrier protein BSA to yield an immunogen.

To a 200 μg/ml physiological saline solution of the (PDM-3-C₂₀-carboxymethyl ether)-BSA complex thus obtained an equal volume ofFreund's complete adjuvant was added, followed by thoroughemulsification. The resulting emulsion was administeredintraperitoneally and subcutaneously at the back to BALB/c mice (female,20 μg/0.2 ml mouse). Additional immunization was conducted at intervalsof 2 to 3 weeks. The animal showing the maximum serum antibody titer 10days after 3 additional immunizations was intravenously given a solutionof (PDM-3-C₂₀ -carboxymethyl ether)-BSA complex (50 μg/0.1 mlphysiological saline/mouse).

2 Cell fusion

Cell fusion was conducted in accordance with the method described inReference Example 2-3.

3 Selection and cloning of hybridomas

Hybridomas were screened by the ELISA method of Reference Example 1using a microplate coupled with (maytansinol3-α-aminophenylacetate)-HSA, followed by the same procedure as inReference Example 2-4 to yield anti-ANS MoAb-producing hybridomas. Fromthese hybridomas was selected a mouse hybridoma AS6-44.9, which showsstrong binding activity to 9-thiomaytansine, MAY and ANS as well as tothe immunogen PDM-3-C₂₀ -carboxymethyl ether. Immunoglobulin class,subclass and light chain type of the present antibody were determined tobe IgG₁ -λ chain by the Ouchterlony and ELISA methods. The antibody wasfound to be capable of neutralizing the cytotoxicity of ANS.

FIG. 1 shows the antibody dilution curve using ELISA for the culturesupernatant of hybridoma AS6-44.9. FIG. 2 shows the neutralizationactivity curves for the cytotoxicity of ANS (target cell lines: mouseleukemia cell line P388D1 and human leukemia cell line K562).

EXAMPLE 2 Production of anti-ANS-anti-hTfR bispecific hybrid monoclonalantibody

1 Cell fusion

The anti-ANS-antibody-producing hybridoma AS6-44.9, obtained in Example1, and the anti-hTfR-antibody-producing hybridoma 22C6, obtained inReference Example 2, were each incubated at 37° C. for 30 minutes forfluorescent staining in an Iskove-Ham F12 mixed medium containing either0.5 μg/ml FITC for hybridoma AS6-44.9 or 1.5 μg/ml TRITC for hybridoma22C6. After addition of an LSM solution (commercially available fromWako Pure Chemical Industries, Ltd.) and removal of dead cells, thesetwo hybridomas were mixed together at a ratio of 1:1 and subjected tocell fusion using PEG6000.

After incubation for 2 hours at 37° C., the cells were applied to FACS,whereby 25,000 fluorescein-rhodamine double stained cells were sorted.These double-stained cells (10 cells/well) were sown to and cultivatedin a 96-well microplate seeded with 5×10⁵ cells/well mouse thymocytes asfeeder cells, with 10 double-stained cells per well.

2 Selection and cloning of hybrid hybridomas

The culture supernatants in the wells in which cell proliferationoccurred 1 to 2 weeks after cell fusion were subjected to the followingELISA procedure for bispecific antibody assay to determine theirantibody titer. To the (maytansinol 3-α-aminophenylacetate)-HSAsensitized plate prepared in Reference Example 1-1 the subject culturesupernatant of hybrid hybridomas was added, followed by reaction at roomtemperature for 2 hours. After the plate was washed with PBS-Tw, abiotin-labeled anti-mouse IgG-_(K) chain-specific antibody was added,followed by reaction at room temperature for 2 hours. Then, HRP-labeledavidin was added and the plate was washed. The activity of the enzymebound to the solid phase was determined by the method described inReference Example 1-2.

Wells showing a high level of hybrid antibody activity were subjected tocloning by the limiting dilution method, whereby the desiredbispecific-antibody-producing tetraoma ATF1-170 was obtained. FIG. 3shows the antibody dilution curve of the culture supernatant ofATF1-170.

3 Purification of hybrid antibody

5×10⁶ tetraoma cells per mouse were intraperitoneally inoculated to sixBALB/c mice which had been intraperitoneally given 0.5 ml mineral oil.Ascites fluid retention began to occur 14 to 18 days later. The ascitesfluid was collected and subjected to salting-out with 45 to 50%saturated ammonium sulfate to yield an IgG fraction. After dialysisagainst 20 mM PBS (pH 7.5), this fraction was applied to a column ofCellulofine coupled with PDM-3-C₂₀ -ρ-aminobenzyl ether and eluted with0.2M glycine-HCl buffer solution (pH 2.9). The acidically elutedfraction was dialyzed against PBS and then subjected to high performanceliquid chromatography using a column of hydroxyapatite to yield theanti-ANS-anti-hTfR bispecific hybrid antibody of the present invention.

About 7.3 mg of the bispecific antibody was obtained from 20 ml ofascites fluid.

EXAMPLE 3 Isolation of anti-ANS-anti-hTfR-bispecific hybrid monoclonalantibody

Tetraoma ATF1-170 was inoculated to BALB/c mice by the method describedin Example 2-3 to obtain ascites fluid. The collected ascites fluid wassubjected to salting-out with 45 to 50% saturated ammonium sulfate andthen purification by a column of Protein A to yield an IgG fraction.This acidically eluted fraction was dialyzed against a 10 mM potassiumphosphate buffer solution (pH 6.2) and applied to a column ofhydroxyapatite equilibrated with the same buffer solution. Elution wasconducted using a density gradient of from 10 mM to 300 mM of potassiumphosphate buffer solution (pH 6.2) to separate various immunoglobulinspecies. The results are shown in FIG. 4.

In FIG. 4, Peak 1 corresponded to the elution position of anti-ANSantibody AS6-44.9, and Peak 3 to the elution position of anti-hTfRantibody 22C6. The antibody elution fractions of respective peaks wereassayed by the ELISA method for bispecific antibody assay described inExample 2-2. As a result, only Peak 2 showed strong antibody activity;it was demonstrated that the desired bispecific hybrid antibody ATF1-170was eluted in Peak 2.

About 2.2 mg of the bispecific antibody was obtained from 5 ml ofascites fluid by the present method.

EXAMPLE 4 Selective cytotoxicity of anti-ANS-anti-hTfR bispecificmonoclonal antibody

The purified anti-ANS-anti-hTfR bispecific antibody obtained in Example3 was reacted with 5 molar equivalents of ansamitocin at 5° C. for 1hour, and then the reaction mixture was subjected to a column ofSephadex G-25 equilibrated with PBS to collect a complex of ANS with thebispecific antibody.

The ANS-antibody complex solution (0.5 ml/well) was added to a plate towhich human leukemia cells (K562; 1.0×10⁴ cells/0.5 ml/well) or mouseleukemia cells (P388D₁ ; 1.0×10⁴ cells/0.5 ml/well) were seeded andcultivation was conducted at 37° C. for 4 days. After the cultivation,the number of cells was determined by a Coulter counter and thecytotoxicity of the ANS-antibody complex was estimated. The results areshown in FIG. 5.

The ANS-antibody complex exhibited a potent cytotoxic effect againsthuman leukemia cell line K562 which has hTfR and a strong bindingactivity with the bispecific antibody of the present invention, butlittle cytotoxic effect on mouse leukemia cell line P388D₁ which has nohTfR.

EXAMPLE 5 Production of anti-ANS-anti-human renal cell carcinomabispecific hybrid monoclonal antibody

1 Cell fusion

According to the method of Example 2, the anti-ANS-antibody-producinghybridoma AS6-44.9, obtained in Example 1, and the anti-human renal cellcarcinoma antibody-producing hybridoma RCS-1, obtained in ReferenceExample 4, were each incubated in an Iskove-Ham F12 mixed mediumcontaining 0.5 μg/ml FITC for hybridoma AS6-44.9 or 1.5 μg/ml TRITC forhybridoma RCS-1 at 37° C. for 30 minutes for fluorescent staining. Afteraddition of an LSM solution (commercially available from Wako PureChemical Industries, Ltd.) and removal of dead cells, these twohybridomas were mixed together at a ratio of 1:1 and subjected to cellfusion using PEG6000.

After incubation for 2 hours at 37° C., the cells were applied to FACS,whereby 25,000 fluorescein-rhodamine double stained cells were sorted.These double-stained cells (10 cells/well) were sown to and cultivatedin a 96-well microplate seeded with 5×10⁵ cells/well mouse thymocytes asfeeder cells, with 10 double-stained cells per well.

2 Selection and cloning of hybrid hybridomas

The culture supernatants in the wells in which cell proliferationoccurred 1 to 2 weeks after cell fusion were subjected to the followingELISA procedure for bispecific antibody assay to determine theirantibody titer. To a microplate on which renal cancer AM-RC-7 cells (10⁴cells/well) were adsorbed the subject culture supernatant of hybridhybridomas was added, followed by reaction at room temperature for 2hours. After the plate was washed with a culture medium, an HRP-labeled(maytansinol 3-α-aminophenylacetate)-HSA was added, followed by reactionat room temperature for 2 hours. Then, the plate was washed. Theactivity of the enzyme bound to the solid phase was determined by themethod described in Reference Example 1-2.

Wells showing a high level of hybrid antibody activity were subjected tocloning by limiting dilution method, whereby the desiredbispecific-antibody-producing tetraoma RCAS1-488 was obtained. FIG. 6shows the antibody dilution curve of the culture supernatant ofRCAS1-488.

3 Purification of hybrid antibody

5×10⁶ tetraoma cells per mouse were intraperitoneally inoculated to sixBALB/c mice which had been intraperitoneally given 0.5 ml mineral oil.Ascites fluid retention began to occur 14 to 18 days later. The ascitesfluid was collected and subjected to salting-out with 45 to 50%saturated ammonium sulfate to yield an IgG fraction. After dialysisagainst 20 mM PBS (pH 7.5), this fraction was applied to a column ofCellulofine coupled with PDM-3-C₂₀₋ρ -aminobenzyl ether and eluted with0.2M glycine-HCl buffer solution (pH 2.9). The acidically elutedfraction was dialyzed against PBS and then subjected to high performanceliquid chromatography using a column of hydroxyapatite to yield theanti-ANS-anti-human renal cell carcinoma bispecific hybrid antibody ofthe present invention.

About 5.8 mg of the bispecific antibody was obtained from 20 ml ofascites fluid.

What is claimed is:
 1. A pharmaceutical composition comprising aneffective amount of an immunocomplex, said immunocomplex comprising acytotoxic ansamitocin derivative with a hybrid monoclonal antibody, saidhybrid monoclonal antibody having binding affinities both to a cytotoxicansamitocin derivative and a target antigen, wherein the cytotoxicity ofthe ansamitocin derivative is reduced when bound to the antibody, in asuitable pharmaceutically acceptable carrier.
 2. A method of reducingthe number of cells of a susceptible tumor in a mammal which comprisesadministering to said mammal an effective amount of an immunocomplexcomprising a cytotoxic ansamitocin derivative with a hybrid monoclonalantibody, said hybrid monoclonal antibody having binding affinities to acytotoxic ansamitocin derivative and a target antigen, wherein thecytotoxicity of the ansamitocin derivative is reduced when bound to theantibody.